rs-17053 and chlorethylclonidine

1. To illuminate the controversy on alpha 1A- or alpha 1L-adrenoceptor involvement in noradrenaline-mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype-selective alpha 1-adrenoceptor agonists and antagonists under different experimental conditions. 2. The agonist potency order in rat SMA was: A61603 >> SKF89748-A > cirazoline > noradrenaline > ST-587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29-8.80) indicating the involvement of alpha 1L-rather than alpha 1A-adrenoceptors. 3. The putative alpha 1L-adrenoceptor antagonist JTH-601, but not the alpha 1B-adrenoceptor antagonist chloroethylclonidine (10 microM) antagonized noradrenaline-induced contractions of SMA. The potency of the selective alpha 1D-adrenoceptor antagonist BMY 7378 against noradrenaline (pA2 = 6.16 +/- 0.13) and of the selective alpha 1A-adrenoceptor antagonist RS-17053 against noradrenaline (pKB = 8.35 +/- 0.10) and against the selective alpha 1A-adrenoceptor agonist A-61603 (pKB = 8.40 +/- 0.09) were too low to account for alpha 1D- and alpha 1A-adrenoceptor involvement. 4. The potency of RS-17053 (pKB/pA2's = 7.72-8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619. 5. Selective protection of a putative alpha 1A-adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS-17053 (pA2 = 8.25 +/- 0.06 against A61603). 6. Combined concentration-ratio analysis demonstrated that tamsulosin, which does not discriminate between alpha 1A- and alpha 1L-adrenoceptors, and RS-17053 competed for binding at the same site in the SMA. 7. In summary, data obtained in our experiments in rat SMA indicate that the alpha 1-adrenoceptor mediating noradrenaline-induced contraction displays a distinct alpha 1L-adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that alpha 1L-adrenoceptors represent an affinity state of the alpha 1A-adrenoceptor in functional assays. Furthermore, there is no co-existing alpha 1A-adrenoceptor in the SMA.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Clonidine; Cresols; Imidazoles; In Vitro Techniques; Indoles; Male; Mesenteric Arteries; Norepinephrine; Piperazines; Potassium; Prazosin; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Sulfonamides; Tamsulosin; Tetrahydronaphthalenes

Alterations in alpha1-adrenoceptor subtypes in aortas from 12-month-old spontaneously hypertensive rats (SHR) were studied in functional studies and RNase protection assays. The norepinephrine-induced contraction, including maximum response and pD2 values, was not significantly different between the SHR and age-matched Kyoto Wistar (WKY) rats. The pA2 values of the alpha1D-adrenoceptor subtype-selective antagonist BMY7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)dec ane-7,9-dionedihydrochloride) were increased from 8.10 +/- 0.12 in WKY rats to 8.45 +/- 0.13 in SHR (P < 0.05). The pA2 values of the alpha1A-adrenoceptor subtype-selective antagonist RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dim ethyl-1H-indole-3-ethanamine hydrochloride) were reduced from 8.52 +/- 0.20 in WKY rats to 7.82 +/- 0.18 in SHR (P < 0.05), whereas the pA2 values of the alpha1A/alpha1D-adrenoceptor subtype-selective antagonist WB4101 (2-(2,6-dimethoxphenoxyethyl)-aminomethyl-1,4 benzodioxane) were not significantly different between WKY rats and SHR (9.05 +/- 0.22 versus 9.27 +/- 0.15, P > 0.05). Preincubation of preparations in 50 microM chloroethylclonidine for 30 min irreversibly inhibited the norepinephrine-induced response more profoundly in aortas from SHR than in aortas from WKY rats. The results of RNase protection assays showed that mRNAs for alpha1A- and alpha1B-adrenoceptor subtypes were decreased and that mRNA for the alpha1D-adrenoceptor subtype was increased in aortas from SHR compared with WKY rats. The results suggested that the alpha1A-adrenoceptor subtype was decreased and the alpha1D-adrenoceptor subtype was increased in aortas of 12-month-old SHR.

Topics: Adrenergic alpha-Antagonists; Animals; Aorta; Clonidine; Dioxanes; Hypertension; Indoles; Male; Muscle Contraction; Norepinephrine; Piperazines; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Adrenergic, alpha-1; Ribonucleases

In this study, the effects of nine alpha-1 adrenoceptor antagonists [prazosin, WB 4101 (WB), chloroethylclonidine (CEC), 5-methylurapidil (5-MU), BMY 7378 (BMY), MDL 73005EF (MDL73), MDL 72832 (MDL72), RS 17053 (RS) and SK&F 105854 (SKF)] were studied on contractile responses to phenylephrine (PE) of the endothelium-denuded dog aorta in vitro. All antagonists, except CEC, 5-MU and RS, produced concentration-dependent competitive inhibition of contractile responses of the aorta to PE. The rightward shift of the concentration-response curves of PE yielded constant pKB values with increasing antagonist concentrations in most cases allowing a single pooled value to be determined: for prazosin, a pKB of 8.99 +/- 0.11 (n = 20, KB of 1.03 nM); for WB, a pKB of 8.75 +/- 0.08 (n = 23, KB of 1.76 nM); for BMY, a pKB of 7.21 +/- 0.13 (n = 13, KB of 62 nM); for MDL72, a pKB of 7.95 +/- 0.15 (n = 12, KB of 11.2 nM); and for SK&F 105854, a pKB of 5.82 +/- 0.08 (n = 15, KB of 1.52 microM). For MDL73, pKB values decreased with antagonist concentration: 7.88 +/- 0.06 at 10 nM, 7.56 +/- 0.28 at 100 nM and 6.92 +/- 0.18 at 1000 nM, which suggests the presence of more than one receptor subtype. CEC (10 and 100 microM) almost completely inhibited responses to PE; lower concentrations had no significant effect. 5-MU (10-300 nM) and RS (3-300 nM) were ineffective antagonists in this tissue. Because WB, a highly selective alpha-1D and alpha-1A adrenoceptor subtypes inhibitor, blocked PE responses (with less affinity than for alpha-1A adrenoceptors), and 5-MU and RS, which are selective blockers for alpha-1A adrenoceptor, were ineffective, we conclude that alpha-1A adrenoceptors are absent in the dog aorta. The effects of the less selective MDL72 were inconsistent with actions at alpha-1B or alpha-1D adrenoceptors. Although WB shifted the PE concentration-response curve to the right, the abilities of BMY, MDL73 and SKF to inhibit competitively PE contraction were of lower affinity compared with expectations for interaction with alpha-1D adrenoceptors; they are not the predominant subtype. The complete inhibition of PE responses by CEC suggests that the dog aorta contains the alpha-1B adrenoceptor subtype. In immunocytochemical studies of the expression of alpha-1B adrenoceptor, all cells apparently expressed this protein. Moreover, Western blot studies of the microsomal fractions confirmed the presence of alpha-1B adrenoceptors. In the dog aorta, the alpha-1 adrenoceptors predomi

Topics: Animals; Aorta; Clonidine; Dioxanes; Dogs; Dose-Response Relationship, Drug; Immunohistochemistry; Indoles; Phenylephrine; Piperazines; Prazosin; Receptors, Adrenergic, alpha-1; Spiro Compounds

The subtype of alpha1-adrenoceptor mediating the exogenous noradrenaline-induced vasopressor response in perfused rat hind limb was determined by functional measurements and radioligand binding assays. The potencies (pA2 values) of alpha1A-adrenoceptor-selective antagonists, RS-17053 (N-[2-(2-cyclopropylmethoxy-phenoxy) ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), WB 4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4 benzodioxane), 5-methyl-urapidil, and the alpha1D-adrenoceptor-selective antagonist, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4.5]de cane-7,9-dione), to inhibit the noradrenaline-induced vasopressor response determined by Schild plot were 9.47 +/- 0.21, 9.48 +/- 0.19, 8.10 +/- 0.27 and 6.66 +/- 0.14, respectively, with no slope significantly different from unity. The affinities (K(i) values) of these antagonists were determined by displacement of 125I-BE 2254 (2-beta(4-hydroxyphenyl)-ethylaminomethyl)-tetralone) binding from the cloned alpha1a-, alpha1b-, alpha1d-adrenoceptor, stably expressed in human embryonic kidney (HEK) 293 cells. The pA2 values of the above antagonists correlated well with the binding K(i) values only for alphaIA-adrenoceptors (r = 0.93), but not for alpha1B-adrenoceptors (r = 0.51) and alpha1D-adrenoceptors (r = 0.13). The concentration-vasopressor response curve for noradrenaline was not significantly affected by pretreatment with 50 microM chloroethylclonidine for 30 min. The results suggest that only alpha1A-adrenoceptors mediate the noradrenaline-induced vasopressor response in perfused rat hind limb.

Topics: Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Antagonists; Animals; Cell Line; Clonidine; Cloning, Molecular; Dioxanes; Dose-Response Relationship, Drug; Extremities; Humans; Indoles; Norepinephrine; Perfusion; Piperazines; Rats; Receptors, Adrenergic, alpha-1; Recombinant Proteins; Vasoconstriction